Single concentration confirmation of inhibitors of Mdm2/MdmX interaction using a Brca1/Bard1 BiLC Counterscreen assay.

A wild type but attenuated p53 is retained in approximately 50% of human tumors, and reactivation of p53 in such tumors is an attractive chemotherapeutic strategy. p53 activity is restricted in vivo by mdm2 and mdmx, and knockout of either of these proteins is embryonic lethal in a p53-dependent manner (1, 2). Both proteins bind to p53 via a hydrophobic N-terminal pocket and block p53-dependent more ..

A wild type but attenuated p53 is retained in approximately 50% of human tumors, and reactivation of p53 in such tumors is an attractive chemotherapeutic strategy. p53 activity is restricted in vivo by mdm2 and mdmx, and knockout of either of these proteins is embryonic lethal in a p53-dependent manner (1, 2). Both proteins bind to p53 via a hydrophobic N-terminal pocket and block p53-dependent transcription of genes required for tumor suppression. Efforts to reactivate p53 with small molecules have focused on inhibition of the mdm2/p53 interaction, which leads to increased p53 levels and activity. However, recent reports indicate that targeting the mdm2/p53 interaction alone is insufficient to induce apoptosis in some cancer cell lines, particularly in the presence of high levels of mdmx (3-5). The most potent mdm2 antagonists do not effectively block mdmx, due to subtle structural alterations of the mdmx hydrophobic pocket (6). Since mdmx is overexpressed in many human tumors with wild type p53, this presents a barrier to effective p53 activation - additional strategies to antagonize mdm2 and mdmx are warranted.

Mdm2 is a member of the E3 ubiquitin ligase family of proteins, which facilitate the transfer of ubiquitin (Ub) molecules from E2 Ub-conjugating enzymes to target substrates (7, 8). This in turn targets the substrate for proteasome-dependent degradation. The C-terminal RING domain of mdm2 is required for ubiquitination and degradation of p53 (7). Although mdmx also has a RING domain, it has no intrinsic ubiquitin ligase activity (9). However, mdm2 and mdmx can homo- or heterodimerize via their RING domains, with a heterodimer being the more stable complex (10, 11). The RING/RING heterodimerization is thought to create an optimal scaffold for positioning and/or processivity of the E2 Ub conjugating enzyme. Therefore, identification of small molecules that disrupt the mdm2/mdmx interaction will provide an additional strategy for p53 reactivation. Development of a small molecule inhibitor of the mdm2/mdmx interaction will permit reversible inactivation of endogenous complexes, thus making analysis of p53 pathway more tractable. The aims of this proposal will be to identify a novel class of p53 activating compounds.

This counterscreen reporter cell-based assay is based on expression of full-length firefly luciferase from a doxycycline-inducible promoter in p53-null SaOS-2 osteosarcoma cell line. The purpose of this counterscreen assay is to deprioritize compounds that: are toxic to the cells, interfere with the luciferase luminescent readout, or inhibit expression / function of luciferase.

The goal of this assay is to validate hits obtained in "uHTS for identification of Inhibitors of Mdm2/MdmX interaction in luminescent format", AID 485346.

Compounds that demonstrated %Efficacy_Mean of >= 23% at 8 uM concentration are considered non-specific and are excluded from further tests and deprioritized for further development.To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below. Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows: 1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data. a. If outcome of the primary screen is inactive, then the assigned score is 0b. If outcome of the primary screen is inconclusive, then the assigned score is 10c. If outcome of the primary screen is active, then the assigned score is 20d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay